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Children s Hospital s Richard Lee on Studying the Urinary Proteome

Richard Lee
Research fellow
Department of Urology, Children's Hospital, Boston

At A Glance

Name: Richard Lee

Position: Research fellow, Department of Urology, Children's Hospital, Boston, since 2004.

Background: Chief resident, Department of Urology, University of Washington, Seattle, 2003-2004. Resident, 2000-2003.

Research fellow, Program in Transplantation Biology, Clinical Research Division, Fred Hutchinson Cancer Research Center, 2001-2002.

Resident, Department of General Surgery, University of Washington, Seattle, 1999.

MD, Jefferson Medical College, 1998.

Last week, Richard Lee gave a talk on urinary proteomics at a symposium to mark the opening of the Children's Hopsital Boston's new Proteomics Center. ProteoMonitor spoke with Lee to find out more about his research, and about the new center.

How did you get into investigating the urinary proteome?

I'm a urologist by training. I'm a urologic surgeon. So we obviously work with a lot of urine and we also work with diseases of the kidney, bladder, prostate, and, in general, the urinary tract. In particular, I specialize in pediatric urology, which deals with many congenital anomalies of the lower urinary tract. And we thought the urine would be an excellent source, because of the proximal fluid nature of urine, for diseases and the potential to find biomarkers in urine for our diseases of interest.

When did you start doing biomarker research?

I started in July of 2004, when I first arrived here. I worked in collaboration with Hanno Steen, [the director of the new Proteomics Center at Children's Hospital Boston], starting off-and-on probably around October of 2004, and then more full-time by March of 2005.

What kind of technologies were you using?

Initially, prior to this center starting, I was mostly using gel-based techniques — spot picking, and then MALDI-TOF analysis.

Subsequently, upon the arrival of Hanno Steen, and the beginning of the set up of the proteomics center, which was initially housed in the urology space, we moved on from there to do direct in-solution digestion of urine samples, and loading those directly on the nano-electrospray LC-MS/MS, and also using the LTQ-FT.

Were you using any pre-fractionation?

We decided not to do any pre-fractionation beforehand in an effort to drill down into the urinary proteome directly from the urine as an initial start to see where we could go from there.

Currently, we've developed an animal model — a rat. The reason why we chose the rat was because obviously we can control a lot of environmental and physiological and genetic variation that we can't limit in humans. We wanted to create a normal control to try to look at normal urine first.

And also, fortunately for us, the rat kidney undergoes significant post-natal maturation after birth — in the first 10 to 14 days of life it completes maturation of its kidney. So as a urologist, and a pediatric urologist in particular, for us, it made much more sense to look at that animal model as a question of, 'What happens to the urinary proteome during normal development?'

So we decided not to pre-fractionate because we didn't know what we would find. We wanted to look first at as much of everything as possible, even the high-abundance proteins. So we have done a comparative analysis from day one of life to adulthood, essentially. In the future we're thinking about fractionating, and multiple different methods to increase the depth of our identifications.

So are you doing mostly cataloguing of proteins in the various stages of development?

Correct. Essentially, yes. We are also currently doing quantitative analysis. We're fractionating to look at different subproteomes within the urine — for instance the phosphoproteome and glycoproteome. And we also have some disease models that we've created that we're looking at.

What have you found in your work with the [controls]?

We've found that there is significant variation from the neonate to the adult. We find that there are unique sets of proteins to just the neonate and the adult that distinguish the two proteomes definitively between each other.

In addition, we hypothesize that some of the proteins we're identifying in the urine early on — that's up to the first 14 days of life — may be representing overall kidney maturation. So they may be markers of kidney maturation.

Do you think this research could be applicable to humans?

We're in the process of trying to validate this in a human model. Obviously we can't get fetal urine, but we have an IRB in place to try to look at normal human urine in neonates, infants, adolescents, and adults, to see if the normals are different.

We want to do this because in our estimation, in order to appropriately try to identify biomarkers, you really need to have a good handle of what is normal first.

What kind of applications do you think there will be once you find out what proteins are representative of what stage of development?

The developmental thing — we did that because we needed a control so that we could look at our disease model in neonatal rats. Particularly, what we're looking at is renal obstruction — when the kidney is unable to send the urine down to the bladder because it essentially is blocked.

That's the first application that we're going to try to find — we're going to try to identify markers in urine that would assist us in determining which children who were born with dilation of the kidney require surgery. We estimate that approximately 2 to 5 percent of all children who are screened with a prenatal ultrasound have dilation of the kidney. That's quite a high number of kids.

There are varying opinions on what is normal and not normal. Current diagnostic tests are not always able to answer that question, so a marker for actual real obstruction may be very beneficial.

What are your disease models?

For renal obstruction, we surgically create blockages of the kidney, or a renal obstruction, in both the neonate and adult [rat] — that's one of the models that we're looking at.

We're using nanoelectrospray LC-MS/MS and also the LTQ-FT to do analysis. We're no longer using 2D gels. We may still do 1D gels as a pre-fractionation method.

We're also trying to look into that as a technical aspect in yield — what are the different methods that we can use to improve our yield? What are the ways we can dig deeper into the proteome? And when we dig deeper, are we finding anything of use?

How will the new center help you with your research?

Without the new center, none of this would be possible, or it would be very difficult. One of the major advantages of having a center here at Children's Hospital is that it is associated with a hospital. More translational research can be performed that may directly relate to more clinical questions.

What's your future direction, now that you have access to this center?

One of our most important future directions is in sample handling and the creation of a urine bank for both normal children and children with diseases. We're really big on trying to get proper sample handling and storage for future use.

We have other animal models that we want to look at. We feel that our initial analysis of the normal urinary proteome of the developing rat has established a nice starting platform for us to take a deeper look at the urinary proteome, and at other diseases.

There are specific urologic childhood diseases that we want to look at in addition to the renal obstruction. For example, something called vesicoureteral reflux. With that disease, when the child voids, the urine, instead of exiting the bladder, goes back towards the kidney. If children get infected, it can cause serious damage to the kidney.

It's a very common disease. It can affect almost 10 percent of children. The good thing is that some children do very well with it and never develop a problem. Other children do horribly with it and need to have surgical correction sooner. They usually can outgrow it, but some kids don't outgrow it, or while they're outgrowing it get multiple infections.

So our goal would be to try to stratify those children through urinary proteomics.

Are there particular challenges to working with the urine, as opposed to other bodily fluids?

Salt. High salt content is a difficulty. The variable pH can be a difficulty. One of the other disadvantages is there may be a lot of temporal variation in protein expression from one person alone — meaning that something collected in the morning is going to be very different from something collected in the afternoon, or in the evening, from the same person.

It's unclear, as with serum, what exactly is normal.

The advantages are that it's very abundant, it's easy to collect — not invasive — and that in general, with most normally functioning kidneys, it does have selective chromatography or fractionation that eliminates all of the high-abundance proteins.

The other advantage is that, in particular for studying disease of the kidney, or the urinary tract, including the prostate, it is the proximal fluid for all of those organs, which is great for the initial biomarker discovery phase.

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